https://orcid.org/0000-0001-7760-4943
Figures
Abstract
The mechanisms of Bisphenol A (BPA) induced learning and memory impairment have still not been fully elucidated. MicroRNAs (miRNAs) are endogenous non-coding small RNA molecules involved in the process of toxicant-induced neurotoxicity. To investigate the role of miRNAs in BPA-induced learning and memory impairment, we analyzed the impacts of BPA on miRNA expression profile by high-throughput sequencing in mice hippocampus. Results showed that mice treated with BPA displayed impairments of spatial learning and memory and changes in the expression of miRNAs in the hippocampus. Seventeen miRNAs were significantly differentially expressed after BPA exposure, of these, 13 and 4 miRNAs were up- and downregulated, respectively. Bioinformatic analysis of Gene Ontology (GO) and pathway suggests that BPA exposure significantly triggered transcriptional changes of miRNAs associated with learning and memory; the top five affected pathways involved in impairment of learning and memory are: 1) Long-term depression (LTD); 2) Thyroid hormone synthesis; 3) GnRH signaling pathway; 4) Long-term potentiation (LTP); 5) Serotonergic synapse. Eight BPA-responsive differentially expressed miRNAs regulating LTP and LTD were further screened to validate the miRNA sequencing data using Real-Time PCR. The deregulation expression levels of proteins of five target genes (CaMKII, MEK1/2, IP3R, AMPAR1 and PLCβ4) were investigated via western blot, for further verifying the results of gene target analysis. Our results showed that LTP and LTD related miRNAs and their targets could contribute to BPA-induced impairment of learning and memory. This study provides valuable information for novel miRNA biomarkers to detect changes in impairment of learning and memory induced by BPA exposure.
Citation: Luo M, Li L, Ding M, Niu Y, Xu X, Shi X, et al. (2023) Long-term potentiation and depression regulatory microRNAs were highlighted in Bisphenol A induced learning and memory impairment by microRNA sequencing and bioinformatics analysis. PLoS ONE 18(1): e0279029. https://doi.org/10.1371/journal.pone.0279029
Editor: Alexandre Hiroaki Kihara, Universidade Federal do ABC, BRAZIL
Received: March 15, 2022; Accepted: November 28, 2022; Published: January 19, 2023
Copyright: © 2023 Luo et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Data Availability: All relevant data are within the manuscript and its Supporting Information files.
Funding: This work was supported by the National Natural Science Foundation of China though a grant awarded to FP (No. 81773402). This work was also supported by the Liaoning Provincial Science and Technology Department Project through a grant awarded to XX (No.2021JH1/10400051).
Competing interests: The authors have declared that no competing interests exist.
Introduction
Bisphenol A (BPA) is an important industrial chemical used extensively worldwide in the production of polycarbonate plastics and epoxy resins [1]. In 2015, the global volume consumption of BPA was estimated at 7.7 million metric tons, and could reach 10.6 million metric tons by 2022 with a compound annual growth rate of 4.8% [2–4]. BPA can seep into our foods and the environment during the manufacturing process or daily use [5]. Humans might be exposed to BPA from fetal to adult stages in various ways including water, air, soil environment, food contamination, etc [6]. The exposure routes of BPA could be oral intake, inhalation or dermal contact [7, 8]. A certain amount of studies performed in cellular cultures, rodents, and humans suggest that BPA overexposure exerts deleterious effects by different mechanisms [9]. BPA works as an agonist on estrogen receptors and antagonist on androgen receptors due to its capability to bind classical nuclear or genomic estrogen receptors [10, 11]. BPA can impair male reproductive function via damaging sperm DNA, decreasing testosterone levels and reducing semen quality [12]. In addition, several studies demonstrated that BPA exposure plays adverse health effects on liver function, increases risk of cardiovascular disease, affects glucose metabolism, disturbs immune function and induces several tumors through binding different receptors, modulating transcription factors, or inducing epigenetic changes [13–16]. It was also found that BPA exposure during embryonic and infant periods may exert toxic impacts on a series of physiological processes such as development on nervous system and brain morphology [17, 18]. In addition, increasing evidence indicated that neurotoxic effects of children/adolescent animals could be induced by BPA exposure [19, 20]. Therefore, more studies on the alterations of behaviors due to the children/adolescent BPA exposure and the involved mechanisms are necessary.
Increasing evidence suggested that epigenetic alterations, including changes in histone modifications, DNA methylation and the expression of non-coding RNAs could be crucial regulators in the long-term effects of environmental toxicants [21, 22]. MicroRNAs (miRNAs), a short non-coding RNAs, regulate translation by binding to the 3′ untranslated region (UTR) of mRNAs [23]. miRNAs play crucial roles in almost all fundamental biological and metabolic processes [24]. Of all the mammalian miRNAs identified to date, more than 50% of them are expressed in the brain [25, 26]. Recent studies have addressed miRNAs to be involved in various central nervous system’s pathologies [27, 28]. Specifically, miRNAs play important roles in the development and progression of neurotoxicity induced by environment stress [29, 30]. Butler et al. reported that miRNA expression change in brain was critical in impairment of BPA-related changes in social-communication behaviors [31]. Kaur et al. demonstrated that altered miRNA levels were associated with BPA-induced anxiety and stereotypical behaviors [32].
Given the considering that miRNAs expression alteration is an important mediator of the BPA-induced impairment of learning and memory, in this study, we exposed young male mice to BPA in drinking water for 8 weeks and investigated the impact of BPA exposure on the miRNA expression profile of the hippocampus. The DIANA-miRPath v3.0 database was used for bioinformatic analysis. Real-Time PCR and western blot were performed for validation of miRNA expression and target genes’ protein levels. Our data would be valuable for revealing novel miRNA biomarkers and possible mechanisms related to impairment of learning and memory induced by BPA exposure.
Methods and materials
Animal handling and procedures used in this study were approved by the Institutional Animal Care and Use Committee of Dalian Medical University (AEE21022). Housing of animals was in agreement with the guidelines of the Animal Care and Use Committee of Dalian Medical University. All procedures were performed under strict accordance with National Institute of Health Guide for Care and Use of Laboratory Animals, and all efforts were made to minimize suffering.
The treatment of animals
Eighty Kunming (KM) mice (Male, aged 4 weeks) weighing 22±2 g were obtained from the Experimental Animal Center of Dalian Medical University. They were housed five per cage under standard conditions, with a 12 h dark-light cycle (lights on at 7:00 a.m.) at 18–22°C and 50% humidity and were maintained on a standard diet with water available ad libitum. All mice were randomly assigned to four groups (n = 20 in each group), including the control group and three BPA exposure groups, according to their body weight. Mice in the four groups were received the following dosages of BPA in the drinking water: 0, 0.05, 0.5, and 5 mg/kg body weight. All treatments were continued for 8 weeks. The volumes of water consumed were measured every 2 days, the weights of the mice were measured every 1 week, and there were no significant differences in water consumption and the body weights of the mice between the BPA-exposed and control groups (Fig 1).
Body weight (a) and water consumption (b) of mice (n = 20). Results are presented as mean ± SEM.
After 8 weeks of BPA treatment, the test of learning and memory ability was performed in all mice from each group (n = 15–20 in each group). Then, mice were euthanized and hippocampus tissues were collected. The hippocampus tissues of three mice in control group and 5mg/mg BPA group were used to construct miRNA expression profile by miRNA sequencing, the hippocampus tissues of other six mice in each group were used to Real-Time PCR and western blot analysis.
The concentrations of BPA exposure were set based on the Reference Dose (RfD) set by the Environmental Protection Agency (0.05 mg/kg body weight, the low BPA-exposed group), 10 times the RfD (0.5 mg/kg body weight, the medium BPA-exposed group) and 100 times the RfD (5 mg/kg, the high BPA-exposed group) [33].
Tests of animal learning and memory ability
Mice from each group were tested for their learning and memory ability using the MWM test as described in our previous research [34]. The device is a circular pool with a diameter of 100 cm, and is filled with water at 23 ± 2°C (depth of water: 40 cm). An escape platform (diameter: 10 cm) was submerged 1 cm below the water surface, was placed in the middle of one quadrant (target quadrant) during the spatial navigation test. Spatial visual cues were black and white geometric figures including triangle, square, circle and diamond placed around the pool in each quadrant. All tests were performed between 15:00 and 22:00, and the water was changed to fresh water daily. The water maze was surrounded by shading curtains for avoiding the interference of light on image acquisition. Briefly, the test included the spatial acquisition phase and probe trial [35, 36]. In spatial acquisition phase, mice were trained for 5 consecutive days. Daily training was starting at different quadrants of the pool in a predetermined pseudorandom order. The time spent to reach the platform (escape latency) within 60 s was recorded as acquisition latency. If the mouse failed to reach the platform within 60 s, it was gently guided onto the platform for 30 s and assigned a latency of 60 s. The escape latency to identify the hidden platform was measured.
On the sixth day, the mice were given a spatial probe test, in which the platform was taken away, and each mouse was placed at one point. The mouse was allowed to navigate freely in the pool for 60 s. The time of swimming in the target quadrant, which had previously contained the hidden platform, was recorded as an indicator of memory retention. The swim paths of mice were recorded and the crossings in the target quadrant were calculated by a smart video tracing system (NoldusEtho Vision system, version 5, Everett, WA, USA).
miRNA library construction and sequencing
Total RNA was extracted from hippocampal tissues by using RNAiso Plus according to the manufacturer’s instruction (Takara, Japan) and then quantified with the NanoPhotometer® spectrophotometer (IMPLEN, CA, USA). Only RNA samples with an A260/A280 of 1.8–2.2 were employed for reverse transcription. RNA concentration was measured using Qubit® RNA Assay Kit in Qubit® 2.0 Flurometer (Life Technologies, CA, USA). RNA integrity was assessed using the RNA Nano 6000 Assay Kit of the Agilent Bioanalyzer 2100 system (Agilent Technologies, CA, USA). 3 μg total RNA per sample was used for the small RNA library. Sequencing libraries were generated with NEBNext® Multiplex Small RNA Library Prep Set for Illumina® (NEB, USA) following manufacturer’s recommendations. The quality of library was evaluated by the Agilent Bioanalyzer 2100 system using DNA High Sensitivity Chips. The clustering of the index-coded samples was performed on a cBot Cluster Generation System using HiSeq Rapid Duo cBot Sample Loading Kit (Illumia) according to the manufacturer’s instructions. After cluster generation, the library preparations were sequenced on an Illumina Hiseq 2500 platform and 50 bp single-end reads were generated.
Bioinformatic evaluation
After miRNA reads were counted and normalized, fold change (FC) between the control and BPA-treated mice was calculated by R program (V3.6.2). The genes whose sum of readcount values of the two groups was less than 10 were filtered out. We selected the differentially expressed miRNAs according to the log2 (FC) and p value threshold. |log2 (FC) |≥ 1 and p value < 0.05 was considered as significant difference. The scatter map and clustering heat map of differential expression miRNAs were presented by R program (V3.6.2) and Python program.
After the differentially expressed miRNAs were screened out, bioinformatic evaluation was carried out for identifying potential functions and prediction of target genes as described previously [30]. The functional enrichment analysis was carried out by DIANA-miRPath v3.0. The Gene Ontology (GO) terms and KEGG pathway analysis were provided to obtain useful information regarding the functions of the altered miRNAs [37–39]. The p-value reflects the significance of GO term enrichment and the pathways correlated to the conditions (The threshold of p-values corrected by false discovery rate (FDR) is 0.05).
The biological significance of altered miRNA expression is intimately associated with their gene targets. Potential target genes of all the differentially expressed miRNAs were predicted from data in the databases: miRDB, miRanda, miRWalk, TargetScan, DIANA-mirPath, and miRNA.org. Then, the results intersected from at least two different programs were retained as the final set of target genes.
Real-Time PCR
One microgram of total RNA was reverse transcribed to cDNA using miRNA Reverse Transcription reagent (Takara, Japan). Quantitative Real-Time PCR was performed with a SYBR Green PCR kit (Takara, Japan) using the TP800 Real-Time PCR Detection System (Takara, Japan). The Bulge-Loop miRNA primers for the selected miRNAs and U6 miRNA were designed and purchased from RiboBio (Guangzhou, China). The U6 small nuclear RNA was used as endogenous control. The primers for the selected genes are shown in S1 Table. The reaction conditions were set as follows: initial denaturation at 94°C for 2 min, followed by 5 cycles of 94°C for 30 s, 55°C for 30 s and 60°C for 30 s. The data were analyzed using the 2−△△CT method.
Western blot analysis
Western blot analysis was performed to detect the protein expression of CaMKII, MEK1/2, AMPAR1, IP3R and PLCβ4. GAPDH was used as a control. Briefly, samples were homogenized in ice-cold RIPA Tissue Protein Extraction Reagent (Biyuntian, China) supplemented with 1% proteinase inhibitor mix and incubated at 4°C for 1h. After incubation, debris was removed via centrifugation at 12,000×g for 15 min at 4°C, and the lysates were stored at -80°C until being used. The total protein concentration in the lysates was determined using a BCA protein assay kit (Biyuntian, China). The samples employed for western blot contained 50 μg of protein from tissues in each lane. The proteins were mixed with an equal volume of SDS-PAGE loading buffer, separated via SDS-PAGE under non-reducing conditions using 10% SDS-PAGE gels and electrotransferred to Hybond-P polyvinylidene fluoride membranes (Millipore, France). The membranes were blocked with blocking buffer containing defatted milk powder for 1 h and incubated overnight at 4°C with 1μg/ml of anti-rabbit CaMKII (1:1000, Abcam, catalog #ab181052), MEK1/2 (1:1000, Abcam, catalog #ab178876), IP3R (1:1000, Abcam, catalog #ab264281), AMPAR1 (1:1000, Cell signaling Technology, catalog #13185S) and PLCβ4 (1:1000, Absin, catalog #abs132943) antibodies. The membrane was washed three times with Tris-buffered saline containing 0.05% Tween-20 (TBST) for 15 min and then incubated at room temperature for 1 h with horseradish peroxidase-conjugated goat anti-rabbit IgG (1:3,000, Sigma-Aldrich, catalog #A0545). The resultant signals were visualized using an enhanced ECL chemiluminescence kit and quantified densitometrically using a UVP BioSpectrum Multispectral Imaging System (Ultra-Violet Products Ltd, Upland, CA). Finally, the band intensity of the target proteins was read using ImageJ software (Bethesda, MD, U.S.A.) against the control sample, GAPDH staining was performed in stripped membranes used to identify the target protein’s antibodies, and normalization was performed by the ratio from band intensity of target proteins against GAPDH.
Statistical analysis
Prior to analyses, we confirmed data normality and homogeneity of variances by D’Agostino–Pearson and Barlett’s tests, respectively. Difference among each group was analyzed by one-way analysis of variance (ANOVA), followed by the Tukey’s multiple-comparison tests. All data set fellows a normal distribution, and are presented as the mean ± standard error of mean (SEM). In all instances a p-value of less than 0.05 was considered to be statistically significant. All data were analyzed with SPSS 23.0 for Windows.
Results
BPA induces impairment of learning and memory
Though MWM test, we found that BPA exposure impaired the mice learning and memory abilities. The representative swimming routes of each group during the spatial acquisition phase (the 5th day) and probe trial were shown (Fig 2A). In the spatial acquisition phase, no significant differences were observed in 0.05 and 0.5 mg/kg BPA-treated mice compared with the control group. 5 mg/kg BPA-treated mice spent a longer time to find the hidden platform than the control mice from the second day (Fig 2B). In the probe test, compared with the control group, the mice in 5 mg/kg BPA-exposed group crossed the platform fewer times (Fig 2C), and stayed shorter in the target quadrant (Fig 2D).
(A) Representative swimming routes of each group during the spatial acquisition phase (the 5th day) and probe trial in the MWM. The representative swimming routes belong to the same animal in each group. In spatial acquisition phase, the representative swimming routes of trail 1–3 are the paths for finding the platform in target quadrant from quadrant I, II or III, respectively. In the probe trial, the representative swimming routes of trail 4 are from the quadrant II to quadrant IV for navigating freely 60 s. (B) In spatial acquisition phase, the time spent to find the hidden platform of mice after exposure to BPA is shown. The data presented is the daily average of trainings in four different quadrants. In the probe trial, the total number of crossings over the platform (C) and the time spent (D) in the target quadrant of mice after exposure to BPA were shown. Results are presented as mean ± SEM. *P < 0.05, **P <0.01 compared with control group.
BPA promotes an alteration of miRNA expression profile in the mice hippocampus
Since BPA exposure at 5 mg/kg impairs learning and memory ability of mice, we investigated the possible expression alterations of miRNAs between the BPA treatment and the control groups in mouse hippocampus. We screened out miRNAs whose relative expression levels changed more than 2 fold between the BPA-treated and control groups. In Fig 3, it was shown that the expression of 17 miRNAs were significantly changed after the BPA treatment (p < 0.05, fold change ≥ 2), of these, 13 were upregulated and 4 were downregulated (Table 1). Variations in the expression of miRNAs between the BPA and control group was exhibited with the clustering heatmap (Fig 4).
Blue dots represent miRNAs with no differential expression; Red and green dots indicate up and downregulation of miRNA, respectively, relative to the control (log2-scaled, p < 0.05).
Red represents high relative expression, and blue represents low relative expression.
Potential signaling pathways for BPA-affected dysregulated miRNA
To gain a better understanding about the function of the differentially expressed miRNAs, the Gene Ontology annotations and their molecular pathways were summarized via mirPath v.3 analysis [30]. The top 10 significantly changed GO terms (GOTERM BP FAT) biological process were highlighted in Table 2. The term “learning and memory” (p = 1.1E-4) showed the significant change.
Furthermore, 8 KEGG pathways were significantly enriched by the dysregulated miRNAs (Table 3): including Long-term depression (LTD), Thyroid hormone synthesis, GnRH signaling pathway, Long-term potentiation (LTP), Serotonergic synapse, Neurotrophin signaling pathway, Glutamatergic synapse, MAPK Signaling Pathway.
BPA exposure alters expression of LTP and LTD related miRNAs
As the top significantly affected pathway after BPA exposure, LTD is a long-lasting decrease in synaptic strength, together with long-lasting increase known as LTP, play crucial roles in the cellular and molecular mechanisms by which memories are formed and stored [40, 41]. Therefore, we focused on the miRNAs related to LTP and LTD.
In Table 4, it was shown that 8 differentially expressed miRNAs were associated with LTP, and 7 were related to LTD. Seven miRNAs play roles in both LTP and LTD, including miR-24-3p, miR-182-5p, miR-96-5p, miR-183-5p, miR-193a-3p, miR-125a-3p and miR-10b-3p; miR-10b-5p only related to LTP. After BPA exposure, miR-10b-3p, miR-182-5p, miR-96-5p, miR-183-5p, miR-10b-5p and miR-125a-3p were upregulated, whereas miR-24-3p and miR-193a-3p were downregulated in BPA group compared with control. We developed Real-Time PCR to confirm the results of miRNA sequencing, the expression levels of these 8 miRNAs displayed a similar regulation trend to the gene sequencing results (Fig 5).
The results are expressed as mean ± SEM (n = 6). *p < 0.05, **p < 0.01, ***p < 0.001 vs the control group.
BPA affected regulation of LTP and LTD regulatory miRNAs’ targets involved in neurotoxicity
We set out to further investigate the function of these 8 LTP and LTD regulatory miRNAs by miRNAs-target predictional algorithms, including miRanda, miRWalk, miRDB, TargetScan, miRNA.org and DIANA-mirPath. For minimizing the number of putative and maybe false positive targets, we select intersections from at least two different databases as miRNA’s targets [30, 42]. In Table 5, it was shown the detailed information of LTP and LTD regulatory target genes of differentially expressed miRNAs.
Full name of miRNAs’ target genes shown in S2 Table.
The functional regulation pathway from BPA exposure to impairment of learning and memory was summarized using LTP and LTD-related miRNAs and their predicted target genes in Fig 6. Some miRNAs can work cooperatively on regulating the target expression. Several miRNAs might regulate more than one target gene in LTP and LTD.
The pathway is obtained from KEGG pathway and confirmed through NCBI PubMed. The target genes involved in LTP and LTD were predicted with at least two of the following databases: miRDB, miRanda, miRWalk, TargetScan, DIANA-mirPath, and miRNA.org. ER: endoplasmic reticulum; AMPAR: glutamate ionotropic α-amino-3-hydroxy-5-methylisoxazole-4-propionic acid receptor; CaMKII: Calcium/calmodulin-dependent protein kinase II; CaMKIV: Calcium/calmodulin-dependent protein kinase IV; CREB: cAMP responsive element binding protei; ERK1/2: extracellular signal-regulated kinase; G: guanine nucleotide binding protein (G protein); Gq: guanine nucleotide binding protein q polypeptide; IP3R: Inositol 1,4,5-triphosphate receptor; MEK1/2: mitogen-activated protein kinase; mGLUR: metabotropic glutamate receptor; NMDAR: N-methyl-D-aspartate receptor; PLCβ4: Phospholipase Cβ4; PLA2: Phospholipase A2; PKC: protein kinase C; Rap1: RAS related protein 1; PKA: protein kinase A; RSK: ribosomal protein S6 kinase.
The confirmation of LTP and LTD regulatory miRNAs’ targets
CaMKII, MEK1/2, IP3R, AMPAR1 and PLCβ4 are important proteins in LTP and LTD, the protein levels of them were shown in Fig 7. Compared to the control group, all analyzed protein levels were decreased in the hippocampus of mice exposed to 5 mg/kg BPA; protein levels of CaMKII, MEK1/2, AMPAR1 and PLCβ4 were decreased in group exposed to 0.5 mg/kg BPA; however, protein levels have no significant changes in group exposed to 0.05 mg/kg BPA.
The results are expressed as mean ± SEM (n = 6). *p < 0.05, **p < 0.01, ***p < 0.001 vs the control group.
Discussion
As an endocrine-disrupting chemical, BPA crosses the blood-brain barriers due to its lipophilicity [3] and has suspected roles as a neuroxicant. BPA-induced neurotoxicity has been correlated with enhancement of a neuroinflammatory conditions and oxidative stress, disruption of axon guidance, and other critical cellular processes [43–45]. Although many studies have shown that some miRNAs may contribute to BPA pathological effects [46], their function in BPA induced impairment on learning and memory ability remains to be explored.
In our present study, a brain region involved in learning and memory, hippocampus was selected for analysis the miRNAs expression after BPA exposure. We found that 17 miRNAs whose expression was changed in mice hippocampus by BPA. Bioinformatics analysis was conducted to further study the potential roles of the target genes regulated by these deregulated miRNAs [47]. GO analysis suggested that target genes of these miRNAs were closely associated with regulation of central nervous system structure and function, including several cognitive processes, especially the learning and memory. KEGG pathway analysis revealed the highly enriched learning and memory-associated pathways, interestingly, the top major pathway affected was LTD. It is widely LTP and LTD are two forms of synaptic plasticity in excitatory neurons [48, 49], known as crucial regulators of long-lasting increase and decrease in synaptic strength [49]. Protein synthesis is required for LTP and LTD [50, 51]. Even transcriptional regulation have largely accounted for the molecular mechanisms underlying characteristic changes in long-lasting synaptic plasticity, modulation of mRNA translation attracted more attention and supporting [50]. miRNAs are important small noncoding RNA molecules, they play important regulatory roles in repression of mRNA translation through binding target sites [52]. Our present study demonstrated that levels of 8 miRNAs related to LTP and LTD were affected by BPA treatment, including miR-10b-3p, miR-10b-5p, miR-182-5p, miR-24-3p, miR-96-5p, miR-193a-3p, miR-183-5p, and miR-125a-3p.
Most of these 8 regulated miRNAs were found to be involved in neural system function and neurological diseases. miR-10b-5p was strongly over-expressed in prefrontal cortex of patients with Huntington’s disease [53]. miR-182-5p and miR-183-5p have been shown that play a critical role in Attention-deficit/hyperactivity disorder [54]. miR-24-3p and miR-495-3p were shown to participant in pathogenesis of Parkinson’s and Alzheimer’s disease [55, 56]. Dysfunctions in synaptic plasticity mechanisms can underlie the cognitive deficit in neurological diseases [57, 58]. Several miRNAs like miR-24-3p has been reported changed during LTP, which contributes to long-lasting modification of synaptic function [59]. Previous study found that LTP induction in the CA1 of mice hippocampus was impaired by environmentally low-dose BPA exposure [19]. BPA significantly modulates LTD in the adult rat hippocampus [60]. Our results might indicate that BPA could impair learning and memory ability through regulating these LTP and LTD related miRNAs.
As certral regulatory factors in epigenetic mechanisms, miRNAs have been proved that mediates excitatory synaptic plasticity via regulating local synaptic protein translation [61]. Protein synthesis and abundance of postsynaptic glutamate receptors, structural and signaling factors is crucial for the maintenance of the change in synaptic strength during LTP [62]. miRNAs can target mRNAs by pairing 3′ untranslated regions, inhibit mRNA stability and translation, regulate synaptic protein synthesis, and control synaptic transmission and plasticity [63]. The study by Stefanovic et al., have reported that miR-125a regulated expression of postsynaptic density 95 (PSD-95) [64]. Lee et al. found that miR-188 served as an important modulator for LTP by negatively targeting neuropilin-2 (Nrp-2) [65]. miR-182 regulated synaptic protein synthesis in long-lasting plasticity through Rac1 [66]. In addition, miR-135 acts by complexin-1/2 to manage the NMDAR induced LTD [67]. Since growing evidence reveal that miRNAs play important roles in the effect of toxicants [68], in our study we found that BPA could disturb the expression of miRNAs, then affect the expression of many crucial signals related to LTP and LTD, and finally induce neurotoxicity.
Interestingly, we noticed that genes encoding NMDAR, AMPAR and mGluRs were identified as targets of multiple miRNAs in LTP and LTD. Molecular events underlying the early phase of LTP / LTD include Ca2+ influx into a neuron through NMDAR and mGluRs, subsequent direct or indirect activation of CaMKII, and CaMKII-dependent insertion of AMPAR into the post-synaptic membrane [69]. A host of protein kinases, such as PLA2, PLCβ4, PKA, PKC, and MAPK, might be triggered and contributed to LTP in various ways. Our bioinformatics analysis showed that these pathways mentioned above may be all affected by BPA.
The CaMKII family consists of four isoforms (alpha, beta, gamma and delta), of which CaMK2A and CaMK2B are highly expressed in the brain and play roles in both hippocampal plasticity. We showed here that BPA could decrease protein level of CaMKII. Our results were consistent with the previous study of Viberg and Lee et al, in which they found that BPA suppressed the activation of CaMKII in mice hippocampus and cerebral cortex of adult male and female mice [70]. As an essential component of MAPK signal transduction pathway, MEK1/2 (MAPK kinase) is involved in many cellular processes such as differentiation, proliferation, transcription regulation, and development [71]. The over-expression of MEK1/2 can selectively activate the ERK1/2 signaling pathway [72, 73], a pathway involved in BPA-induced impairment of synaptic plasticity [74]. PLCβ4 is an important kinase mediate signals from mGluR1 that are crucial for the modulation of synaptic transmission and plasticity. As a downstream signaling pathway of PLCβ4, IP3R is mediating Ca2+ release from the endoplasmic reticulum [75]. The expression of PLCβ4 and IP3R were affected by neurotoxicants [49, 76]. Our work is the first report that expression of PLCβ4 and IP3R can be affected by BPA in brain.
We are finding that the AMPAR is regulating by miR-125a-3p, miR-96-5p, miR-182-5p and miR-24-3p; MEK1/2 and IP3R are the targets of both miR-182-5p and miR-96-5p; CaMKII is regulated by miR-10b-5p and miR-24-3p; PLCβ4 is regulated by miR-96-5p and miR-183-5p. These findings suggest that miRNAs can act cooperatively to regulate target expression in neurons. Our present results indicated that the impairment of learning and memory might be due to, or partly, the results of miRNAs’ co-regulation on LTP and LTD.
In addition, some proteins are involved in the regulation of multiple pathways. Such as MEK1/2 protein, belongs to MAPK family, was also identified as targets of the miRNAs in the other pathways, such as MAPK pathway. In the current study, besides the findings on BPA regulate LTP and LTD pathways, some other significantly differentially enriched pathways such as GnRH signaling pathway, Glutamatergic synapse pathway, Serotonergic synapse pathway, Neurotrophin signaling pathway, Glutamatergic synapse pathway, MAPK pathway might take part in the BPA mediated learning and memory impairment. Therefore, further studies are needed to confirm their function in neurotoxicity.
Conclusion
This is the first study to explore the miRNAs regulation in BPA-induced learning and memory impairment. The results indicate that the differentially expressed miRNAs identified in the hippocampus could be the targets of BPA, which may play function via LTP and LTD. Our present study not only provides new insights into the pathogenesis of BPA especially linking to impairment of learning and memory, but it also provides us clues for future mechanism exploring.
References
- 1. Santoro A, Chianese R, Troisi J, Richards S, Nori SL, Fasano S, et al. Neuro-toxic and Reproductive Effects of BPA. Current neuropharmacology. 2019;17(12):1109–32. Epub 2019/08/01. pmid:31362658; PubMed Central PMCID: PMC7057208.
- 2. Sonavane M, Gassman NR. Bisphenol A co-exposure effects: a key factor in understanding BPA’s complex mechanism and health outcomes. Crit Rev Toxicol. 2019;49(5):371–86. pmid:31256736.
- 3. Wang H, Chang L, Aguilar JS, Dong S, Hong Y. Bisphenol-A exposure induced neurotoxicity in glutamatergic neurons derived from human embryonic stem cells. Environment international. 2019;127:324–32. Epub 2019/04/07. pmid:30953815.
- 4. Research and Markets. The world’s largest market research store 2022 [cited 2022 July 26]. Available from: https://www.researchandmarkets.com/.
- 5. Zhang H, Wang Z, Meng L, Kuang H, Liu J, Lv X, et al. Maternal exposure to environmental bisphenol A impairs the neurons in hippocampus across generations. Toxicology. 2020;432:152393. Epub 2020/02/07. pmid:32027964.
- 6. Pang W, Lian FZ, Leng X, Wang SM, Li YB, Wang ZY, et al. Microarray expression profiling and co-expression network analysis of circulating LncRNAs and mRNAs associated with neurotoxicity induced by BPA. Environmental science and pollution research international. 2018;25(15):15006–18. Epub 2018/03/20. pmid:29552716.
- 7. Chianese R, Troisi J, Richards S, Scafuro M, Fasano S, Guida M, et al. Bisphenol A in Reproduction: Epigenetic Effects. Curr Med Chem. 2018;25(6):748–70. Epub 2017/10/11. pmid:28990514.
- 8. Vandenberg LN, Hauser R, Marcus M, Olea N, Welshons WV. Human exposure to bisphenol A (BPA). Reprod Toxicol. 2007;24(2):139–77. Epub 2007/09/11. pmid:17825522.
- 9. Cimmino I, Fiory F, Perruolo G, Miele C, Beguinot F, Formisano P, et al. Potential Mechanisms of Bisphenol A (BPA) Contributing to Human Disease. International journal of molecular sciences. 2020;21(16). Epub 2020/08/17. pmid:32796699; PubMed Central PMCID: PMC7460848.
- 10. Richter CA, Birnbaum LS, Farabollini F, Newbold RR, Rubin BS, Talsness CE, et al. In vivo effects of bisphenol A in laboratory rodent studies. Reproductive toxicology (Elmsford, NY). 2007;24(2):199–224. Epub 2007/08/09. pmid:17683900; PubMed Central PMCID: PMC2151845.
- 11. Wetherill YB, Akingbemi BT, Kanno J, McLachlan JA, Nadal A, Sonnenschein C, et al. In vitro molecular mechanisms of bisphenol A action. Reproductive toxicology (Elmsford, NY). 2007;24(2):178–98. Epub 2007/07/14. pmid:17628395.
- 12. Meeker JD, Ehrlich S, Toth TL, Wright DL, Calafat AM, Trisini AT, et al. Semen quality and sperm DNA damage in relation to urinary bisphenol A among men from an infertility clinic. Reproductive toxicology (Elmsford, NY). 2010;30(4):532–9. Epub 2010/07/27. pmid:20656017; PubMed Central PMCID: PMC2993767.
- 13. Martella A, Silvestri C, Maradonna F, Gioacchini G, Allarà M, Radaelli G, et al. Bisphenol A Induces Fatty Liver by an Endocannabinoid-Mediated Positive Feedback Loop. Endocrinology. 2016;157(5):1751–63. Epub 2016/03/26. pmid:27014939; PubMed Central PMCID: PMC6285285.
- 14. Provvisiero DP, Pivonello C, Muscogiuri G, Negri M, de Angelis C, Simeoli C, et al. Influence of Bisphenol A on Type 2 Diabetes Mellitus. International journal of environmental research and public health. 2016;13(10). Epub 2016/10/27. pmid:27782064; PubMed Central PMCID: PMC5086728.
- 15. Xu J, Huang G, Guo TL. Developmental Bisphenol A Exposure Modulates Immune-Related Diseases. Toxics. 2016;4(4). Epub 2017/10/21. pmid:29051427; PubMed Central PMCID: PMC5606650.
- 16. Weinhouse C, Anderson OS, Bergin IL, Vandenbergh DJ, Gyekis JP, Dingman MA, et al. Dose-dependent incidence of hepatic tumors in adult mice following perinatal exposure to bisphenol A. Environmental health perspectives. 2014;122(5):485–91. Epub 2014/02/04. pmid:24487385; PubMed Central PMCID: PMC4014767 interests.
- 17. Wolstenholme JT, Rissman EF, Connelly JJ. The role of Bisphenol A in shaping the brain, epigenome and behavior. Hormones and behavior. 2011;59(3):296–305. Epub 2010/10/30. pmid:21029734; PubMed Central PMCID: PMC3725332.
- 18. Wu D, Wu F, Lin R, Meng Y, Wei W, Sun Q, et al. Impairment of learning and memory induced by perinatal exposure to BPA is associated with ERα-mediated alterations of synaptic plasticity and PKC/ERK/CREB signaling pathway in offspring rats. Brain research bulletin. 2020;161:43–54. Epub 2020/05/08. pmid:32380187.
- 19. Zhou Y, Wang Z, Xia M, Zhuang S, Gong X, Pan J, et al. Neurotoxicity of low bisphenol A (BPA) exposure for young male mice: Implications for children exposed to environmental levels of BPA. Environ Pollut. 2017;229:40–8. Epub 2017/06/04. pmid:28577381.
- 20. Hong SB, Hong YC, Kim JW, Park EJ, Shin MS, Kim BN, et al. Bisphenol A in relation to behavior and learning of school-age children. J Child Psychol Psychiatry. 2013;54(8):890–9. Epub 2013/03/01. pmid:23445117.
- 21. Barouki R, Melén E, Herceg Z, Beckers J, Chen J, Karagas M, et al. Epigenetics as a mechanism linking developmental exposures to long-term toxicity. Environ Int. 2018;114:77–86. pmid:29499450.
- 22. McLachlan JA. Environmental signaling: from environmental estrogens to endocrine-disrupting chemicals and beyond. Andrology. 2016;4(4):684–94. Epub 2016/05/28. pmid:27230799.
- 23. Filipowicz W, Bhattacharyya SN, Sonenberg N. Mechanisms of post-transcriptional regulation by microRNAs: are the answers in sight? Nat Rev Genet. 2008;9(2):102–14. Epub 2008/01/17. pmid:18197166.
- 24. Ambros V. The functions of animal microRNAs. Nature. 2004;431(7006):350–5. Epub 2004/09/17. pmid:15372042.
- 25. Kleaveland B, Shi CY, Stefano J, Bartel DP. A Network of Noncoding Regulatory RNAs Acts in the Mammalian Brain. Cell. 2018;174(2):350–62 e17. pmid:29887379.
- 26. Chakraborty S, Islam MR, Ali MM, Nabi A. Evolutionary Divergence of Brain-specific Precursor miRNAs Drives Efficient Processing and Production of Mature miRNAs in Human. Neuroscience. 2018;392:141–59. Epub 2018/10/03. pmid:30273624.
- 27. Cao DD, Li L, Chan WY. MicroRNAs: Key Regulators in the Central Nervous System and Their Implication in Neurological Diseases. International journal of molecular sciences. 2016;17(6). Epub 2016/05/31. pmid:27240359; PubMed Central PMCID: PMC4926376.
- 28. Meza-Sosa KF, Valle-García D, Pedraza-Alva G, Pérez-Martínez L. Role of microRNAs in central nervous system development and pathology. Journal of neuroscience research. 2012;90(1):1–12. Epub 2011/09/17. pmid:21922512.
- 29. Zhan Y, Guo Z, Zheng F, Zhang Z, Li K, Wang Q, et al. Reactive oxygen species regulate miR-17-5p expression via DNA methylation in paraquat-induced nerve cell damage. Environ Toxicol. 2020;35(12):1364–73. Epub 2020/07/22. pmid:32691990.
- 30. Piao F, Chen Y, Yu L, Shi X, Liu X, Jiang L, et al. 2,5-hexanedione-induced deregulation of axon-related microRNA expression in rat nerve tissues. Toxicology letters. 2020;320:95–102. Epub 2019/11/25. pmid:31760062.
- 31. Butler MC, Long CN, Kinkade JA, Green MT, Martin RE, Marshall BL, et al. Endocrine disruption of gene expression and microRNA profiles in hippocampus and hypothalamus of California mice: Association of gene expression changes with behavioural outcomes. J Neuroendocrinol. 2020;32(5):e12847. pmid:32297422.
- 32. Kaur S, Kinkade JA, Green MT, Martin RE, Willemse TE, Bivens NJ, et al. Disruption of global hypothalamic microRNA (miR) profiles and associated behavioral changes in California mice (Peromyscus californicus) developmentally exposed to endocrine disrupting chemicals. Hormones and behavior. 2021;128:104890. Epub 2020/11/23. pmid:33221288; PubMed Central PMCID: PMC7897400.
- 33. United States Environmental Protection Agency. Chemicals under the Toxic Substances Control Act (TSCA) 2012 [updated July 22, 2022; cited 2022 July 26]. Available from: www.epa.gov/oppt/exposure/presentations/efast/usepa1997efh.pdf.
- 34. Wang D, Wang X, Liu X, Jiang L, Yang G, Shi X, et al. Inhibition of miR-219 Alleviates Arsenic-Induced Learning and Memory Impairments and Synaptic Damage Through Up-regulating CaMKII in the Hippocampus. Neurochemical research. 2018;43(4):948–58. Epub 2018/02/27. pmid:29478199.
- 35. Nicholls RE, Alarcon JM, Malleret G, Carroll RC, Grody M, Vronskaya S, et al. Transgenic mice lacking NMDAR-dependent LTD exhibit deficits in behavioral flexibility. Neuron. 2008;58(1):104–17. Epub 2008/04/11. pmid:18400167.
- 36. Bilgic Y, Demir EA, Bilgic N, Dogan H, Tutuk O, Tumer C. Detrimental effects of chia (Salvia hispanica L.) seeds on learning and memory in aluminum chloride‑induced experimental Alzheimer’s disease. Acta Neurobiol Exp (Wars). 2018;78(4):322–31. Epub 2019/01/10. pmid:30624431.
- 37. Ashburner M, Ball CA, Blake JA, Botstein D, Butler H, Cherry JM, et al. Gene ontology: tool for the unification of biology. The Gene Ontology Consortium. Nat Genet. 2000;25(1):25–9. pmid:10802651.
- 38. Humphreys BD. Mechanisms of Renal Fibrosis. Annu Rev Physiol. 2018;80:309–26. Epub 2017/10/27. pmid:29068765.
- 39. Jantzen SG, Sutherland BJ, Minkley DR, Koop BF. GO Trimming: Systematically reducing redundancy in large Gene Ontology datasets. BMC Res Notes. 2011;4:267. pmid:21798041.
- 40. Manabe T. Two forms of hippocampal long-term depression, the counterpart of long-term potentiation. Rev Neurosci. 1997;8(3–4):179–93. Epub 1997/07/01. pmid:9548231.
- 41. Bliss TV, Collingridge GL. A synaptic model of memory: long-term potentiation in the hippocampus. Nature. 1993;361(6407):31–9. Epub 1993/01/07. pmid:8421494.
- 42. Zhang QL, Dong ZX, Xiong Y, Li HW, Guo J, Wang F, et al. Genome-wide transcriptional response of microRNAs to the benzo(a)pyrene stress in amphioxus Branchiostoma belcheri. Chemosphere. 2019;218:205–10. Epub 2018/11/25. pmid:30471501.
- 43. Akintunde JK, Akintola TE, Adenuga GO, Odugbemi ZA, Adetoye RO, Akintunde OG. Naringin attenuates Bisphenol-A mediated neurotoxicity in hypertensive rats by abrogation of cerebral nucleotide depletion, oxidative damage and neuroinflammation. Neurotoxicology. 2020;81:18–33. Epub 2020/08/19. pmid:32810514.
- 44. Pang Q, Li Y, Meng L, Li G, Luo Z, Fan R. Neurotoxicity of BPA, BPS, and BPB for the hippocampal cell line (HT-22): An implication for the replacement of BPA in plastics. Chemosphere. 2019;226:545–52. Epub 2019/04/07. pmid:30953899.
- 45. Nguyen U, Tinsley B, Sen Y, Stein J, Palacios Y, Ceballos A, et al. Exposure to bisphenol A differentially impacts neurodevelopment and behavior in Drosophila melanogaster from distinct genetic backgrounds. Neurotoxicology. 2021;82:146–57. pmid:33309840.
- 46. Farahani M, Rezaei-Tavirani M, Arjmand B. A systematic review of microRNA expression studies with exposure to bisphenol A. Journal of applied toxicology: JAT. 2021;41(1):4–19. Epub 2020/07/15. pmid:32662106.
- 47. Tian T, Zhang Y, Wu T, Yang L, Chen C, Li N, et al. miRNA profiling in the hippocampus of attention-deficit/hyperactivity disorder rats. Journal of cellular biochemistry. 2019;120(3):3621–9. Epub 2018/10/03. pmid:30270454.
- 48. Chen WR, Lee S, Kato K, Spencer DD, Shepherd GM, Williamson A. Long-term modifications of synaptic efficacy in the human inferior and middle temporal cortex. Proc Natl Acad Sci U S A. 1996;93(15):8011–5. pmid:8755594.
- 49. Zhang C, Li S, Sun Y, Dong W, Piao F, Piao Y, et al. Arsenic downregulates gene expression at the postsynaptic density in mouse cerebellum, including genes responsible for long-term potentiation and depression. Toxicology letters. 2014;228(3):260–9. Epub 2014/05/17. pmid:24831965.
- 50. Kelleher RJ 3rd, Govindarajan A, Tonegawa S. Translational regulatory mechanisms in persistent forms of synaptic plasticity. Neuron. 2004;44(1):59–73. Epub 2004/09/29. pmid:15450160.
- 51. Costa-Mattioli M, Sossin WS, Klann E, Sonenberg N. Translational control of long-lasting synaptic plasticity and memory. Neuron. 2009;61(1):10–26. pmid:19146809.
- 52. Bartel DP. MicroRNAs: genomics, biogenesis, mechanism, and function. Cell. 2004;116(2):281–97. Epub 2004/01/28. pmid:14744438.
- 53. Hoss AG, Labadorf A, Latourelle JC, Kartha VK, Hadzi TC, Gusella JF, et al. miR-10b-5p expression in Huntington’s disease brain relates to age of onset and the extent of striatal involvement. BMC Med Genomics. 2015;8:10. pmid:25889241.
- 54. Piña R, Rozas C, Contreras D, Hardy P, Ugarte G, Zeise ML, et al. Atomoxetine Reestablishes Long Term Potentiation in a Mouse Model of Attention Deficit/Hyperactivity Disorder. Neuroscience. 2020;439:268–74. Epub 2019/12/07. pmid:31809728.
- 55. Rahman MR, Islam T, Zaman T, Shahjaman M, Karim MR, Huq F, et al. Identification of molecular signatures and pathways to identify novel therapeutic targets in Alzheimer’s disease: Insights from a systems biomedicine perspective. Genomics. 2020;112(2):1290–9. Epub 2019/08/05. pmid:31377428.
- 56. Uwatoko H, Hama Y, Iwata IT, Shirai S, Matsushima M, Yabe I, et al. Identification of plasma microRNA expression changes in multiple system atrophy and Parkinson’s disease. Mol Brain. 2019;12(1):49. pmid:31088501.
- 57. Li W, Liu H, Yu M, Zhang X, Zhang Y, Liu H, et al. Folic Acid Alters Methylation Profile of JAK-STAT and Long-Term Depression Signaling Pathways in Alzheimer’s Disease Models. Mol Neurobiol. 2016;53(9):6548–56. Epub 2016/11/01. pmid:26627706.
- 58. Minhas PS, Latif-Hernandez A, McReynolds MR, Durairaj AS, Wang Q, Rubin A, et al. Restoring metabolism of myeloid cells reverses cognitive decline in ageing. Nature. 2021;590(7844):122–8. Epub 2021/01/22. pmid:33473210.
- 59. Ryan MM, Ryan B, Kyrke-Smith M, Logan B, Tate WP, Abraham WC, et al. Temporal profiling of gene networks associated with the late phase of long-term potentiation in vivo. PLoS One. 2012;7(7):e40538. pmid:22802965.
- 60. Hasegawa Y, Ogiue-Ikeda M, Tanabe N, Kimoto T, Hojo Y, Yamazaki T, et al. Bisphenol A significantly modulates long-term depression in the hippocampus as observed by multi-electrode system. Neuro Endocrinol Lett. 2013;34(2):129–34. Epub 2013/05/07. pmid:23645310.
- 61. Sambandan S, Akbalik G, Kochen L, Rinne J, Kahlstatt J, Glock C, et al. Activity-dependent spatially localized miRNA maturation in neuronal dendrites. Science (New York, NY). 2017;355(6325):634–7. Epub 2017/02/12. pmid:28183980.
- 62. Biever A, Donlin-Asp PG, Schuman EM. Local translation in neuronal processes. Current opinion in neurobiology. 2019;57:141–8. Epub 2019/03/13. pmid:30861464.
- 63. Schratt G. microRNAs at the synapse. Nature reviews Neuroscience. 2009;10(12):842–9. Epub 2009/11/06. pmid:19888283.
- 64. Stefanovic S, Bassell GJ, Mihailescu MR. G quadruplex RNA structures in PSD-95 mRNA: potential regulators of miR-125a seed binding site accessibility. RNA (New York, NY). 2015;21(1):48–60. Epub 2014/11/20. pmid:25406362; PubMed Central PMCID: PMC4274637.
- 65. Lee K, Kim JH, Kwon OB, An K, Ryu J, Cho K, et al. An activity-regulated microRNA, miR-188, controls dendritic plasticity and synaptic transmission by downregulating neuropilin-2. The Journal of neuroscience: the official journal of the Society for Neuroscience. 2012;32(16):5678–87. Epub 2012/04/20. pmid:22514329; PubMed Central PMCID: PMC5010781.
- 66. Griggs EM, Young EJ, Rumbaugh G, Miller CA. MicroRNA-182 regulates amygdala-dependent memory formation. The Journal of neuroscience: the official journal of the Society for Neuroscience. 2013;33(4):1734–40. Epub 2013/01/25. pmid:23345246; PubMed Central PMCID: PMC3711533.
- 67. Hu Z, Yu D, Gu QH, Yang Y, Tu K, Zhu J, et al. miR-191 and miR-135 are required for long-lasting spine remodelling associated with synaptic long-term depression. Nature communications. 2014;5:3263. Epub 2014/02/19. pmid:24535612; PubMed Central PMCID: PMC3951436.
- 68. Yu HW, Cho WC. The role of microRNAs in toxicology. Archives of toxicology. 2015;89(3):319–25. Epub 2015/01/15. pmid:25586887.
- 69. Duda P, Wójtowicz T, Janczara J, Krowarsch D, Czyrek A, Gizak A, et al. Fructose 1,6-Bisphosphatase 2 Plays a Crucial Role in the Induction and Maintenance of Long-Term Potentiation. Cells. 2020;9(6). Epub 2020/06/05. pmid:32492972; PubMed Central PMCID: PMC7349836.
- 70. Viberg H, Lee I. A single exposure to bisphenol A alters the levels of important neuroproteins in adult male and female mice. Neurotoxicology. 2012;33(5):1390–5. Epub 2012/09/18. pmid:22981971.
- 71. Wooten MW, Seibenhener ML, Neidigh KB, Vandenplas ML. Mapping of atypical protein kinase C within the nerve growth factor signaling cascade: relationship to differentiation and survival of PC12 cells. Mol Cell Biol. 2000;20(13):4494–504. pmid:10848576.
- 72. Zhou C, Li C, Li D, Wang Y, Shao W, You Y, et al. BIG1, a brefeldin A-inhibited guanine nucleotide-exchange protein regulates neurite development via PI3K-AKT and ERK signaling pathways. Neuroscience. 2013;254:361–8. Epub 2013/10/05. pmid:24090963.
- 73. Zhou Y, Pernet V, Hauswirth WW, Di Polo A. Activation of the extracellular signal-regulated kinase 1/2 pathway by AAV gene transfer protects retinal ganglion cells in glaucoma. Mol Ther. 2005;12(3):402–12. Epub 2005/06/25. pmid:15975850.
- 74. Yang Y, Fang Z, Dai Y, Wang Y, Liang Y, Zhong X, et al. Bisphenol-A antagonizes the rapidly modulating effect of DHT on spinogenesis and long-term potentiation of hippocampal neurons. Chemosphere. 2018;195:567–75. Epub 2017/12/27. pmid:29278848.
- 75. Nakamura M, Sato K, Fukaya M, Araishi K, Aiba A, Kano M, et al. Signaling complex formation of phospholipase Cbeta4 with metabotropic glutamate receptor type 1alpha and 1,4,5-trisphosphate receptor at the perisynapse and endoplasmic reticulum in the mouse brain. Eur J Neurosci. 2004;20(11):2929–44. Epub 2004/12/08. pmid:15579147.
- 76. Dusza HM, Cenijn PH, Kamstra JH, Westerink RHS, Leonards PEG, Hamers T. Effects of environmental pollutants on calcium release and uptake by rat cortical microsomes. Neurotoxicology. 2018;69:266–77. Epub 2018/07/30. pmid:30056177.